The field of the invention relates to targeted delivery of nucleic acid. In particular, the field of the invention relates to targeted delivery of nucleic acid such as small inhibitory ribonucleic acid (siRNA) via landscape phage fusion proteins.
The Ff class of filamentous phage includes three strains referred to as fl, fd, and M13. These phages are thread-like particles approximately 1000 nm long and 7 nm in diameter. The majority of the tubular capsid consists of 2700 identical, largely alpha-helical subunits of the ˜50-residue pVIII major coat protein. Viral DNA of varying sizes, including recombinant genomes with foreign DNA inserts, can be accommodated in the filamentous capsid whose length is altered to match the size of the enclosed DNA by adding proportionally fewer or more pVIII subunits during phage assembly. Additional phage coat proteins include pIII, pVI, pVII, and pIX. One tip of the phage outer tube is capped with five copies each of the minor coat proteins pVII and pIX and another tip with the minor coat proteins pIII and pVI.
In phage display constructs and libraries formed therefrom, heterologous or foreign coding sequences are spliced in-frame into one of the five phage coat protein genes, so that the ‘guest’ peptide, encoded by that sequence, is fused to the coat protein to form a fusion protein which displays the guest peptide on the surface of the virion. A phage display library is a collection of such fusion phage clones, each harboring a different foreign coding sequence, and therefore displaying a different guest peptide on their surface. When a foreign coding sequence is spliced into the major coat protein's gene (pVIII), the guest peptide is displayed on every pVIII subunit increasing the virion's total mass by up to 20%. Such particles are generally referred to as “landscape phage” to emphasize the dramatic change in surface architecture caused by arraying thousands of copies of the foreign peptide in a dense, repeating pattern around the viral capsid. A landscape library refers to a large population of such phages, encompassing billions of clones with different surface structures and biophysical properties. Landscape libraries may be screened to identify phage that bind specifically to selected targets such as cancer cells. The fusion proteins isolated from the selected phage can be utilized to target liposomes specifically to these targets. (See U.S. Patent Publication No. U.S. 2007-0077291; and Bedi et al., Nanomedicine 2010 Nov. 2 [Epub ahead of print], PMID:21050894; the contents of which are incorporated herein by reference in their entireties).
Delivery of nucleic acid to target cells holds considerable promise as a therapeutic approach to treating various diseases, for example, via gene therapy or via regulating gene expression. In particular, RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes. Here, a nanotechnological platform for delivering nucleic acid to target cells is proposed. This platform utilizes the unique propensity of phage proteins to self-assemble in the presence of nucleic acids and to form particles mimicking the structure of the phage capsid.